Enhanced extracellular respiration of engineered Bacillus subtilis via anodic electro-fermentation with pH optimisation

Abstract Leveraging alternative electron acceptors to support anaerobic metabolism in industrially relevant microorganisms holds substantial biotechnological potential, especially when coupled to anodic electro-fermentation, which provides a non-depleting electron sink to the microorganisms. Bacillus subtilis is a widely used industrial workhorse for biochemical production, valued for its genetic tractability and environmental stress tolerance. However, the anaerobic, anodic metabolism of B . subtilis has been constrained by limited redox flexibility. Here, deletion of ldh (lactate dehydrogenase) to restrict fermentative NAD⁺ regeneration enabled engineered B. subtilis to survive anaerobically via anodic respiration, partially oxidising glucose while steering the metabolism toward 2,3-butanediol. The anodic metabolism showed enhanced extracellular electron transfer mediated by ferricyanide, with the highest current density of 0.77 mA/cm 2 reached within 2 h. Carbon flux was directed predominantly to 2,3-butanediol (0.49 ± 0.07 mol product /mol glucose ) under incomplete glucose oxidation and without pH control. In addition, pH control further improved anodic electro-fermentation performance. At pH 6.5, 66% of the added glucose was consumed, and 2,3-butanediol carbon selectivity rose to 77.1 ± 0.6%, whereas at pH 7.5, cells consumed 89% of the glucose with 73.4 ± 0.7% 2,3-butanediol carbon selectivity. To our knowledge, this is the first investigation of anodic electro-fermentation in B. subtilis that integrates metabolic engineering with pH optimisation strategies, demonstrating a new route to produce high-purity biochemicals from renewable resources.